How do restriction enzymes digest?

Protocol for DNA Digestion with a Single Restriction Enzyme

  1. Add components to a clean tube in the order shown:
  2. Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
  3. Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA.

What is digestion of DNA?

Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes or RE’s).

Why do we use two restriction enzymes?

Using two different restriction enzyme sites can help ensure the correct orientation of the gene of interest when it is inserted and prevent the plasmid vector from ligating with itself.

How many restriction enzymes are used in digest?

Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of many commonly used restriction enzymes, visit NEB.

Can you do a triple digest?

Yes, triple or quadruple digestions work, as long as the buffer is compatible with all three or four enzymes, and as long as their recognition sites are not too close to each other (as a rule of thumb, they should be separated by at least 4 bp -though you won’t have that problem in your scenario).

How does a double digest differ from a single digest?

In single-digested plasmids, digestion with the same restriction enzyme produce both ends while, in double-digested plasmids, digestion with a different restriction enzyme produce each end. Hence, this is an important main difference between single digested plasmid and double digested plasmid.

Can we digest DNA?

Basically, DNA, like proteins and complex carbohydrates, gets broken down into pieces – this is what digestion is all about. Your teeth mash it up and enzymes throughout your digestive tract cut it to pieces.